The present disclosure relates to a cartridge for biochemical analyses, a system for biochemical analyses, and a method of carrying out a biochemical process.
As is known, analysis of nucleic acids involves, according to different modalities, preliminary steps of preparation of a specimen of biological material, amplification of the nucleic material contained therein, and hybridization of individual target or reference strands, corresponding to the sequences sought. Hybridization occurs (and the test yields a positive result) if the specimen contains strands complementary to the target strands.
At the end of the preparatory steps, the specimen is examined to check whether hybridization has taken place (the so-called “detection step”).
Several inspection methods and apparatuses are known for this purpose, for example of an optical or electrical type. In particular, the methods and apparatuses of an optical type are frequently based upon the phenomenon of fluorescence. The amplification and hybridization reactions are conducted in such a way that the hybridized strands, contained in a detection chamber provided in a support, include fluorescent molecules or fluorophores (the hybridized strands can be fixed to the bottom of the detection chamber or else remain in liquid suspension). The support is exposed to a light source having an appropriate spectrum of emission such as to excite the fluorophores. In turn, the excited fluorophores emit a secondary radiation at a wavelength of emission higher than that of the peak of the excitation spectrum. The light emitted by the fluorophores is collected and detected by means of an optical sensor. In order to eliminate the background light radiation, which represents a source of disturbance, the optical sensor is provided with bandpass filters centred at the wavelength of emission of the fluorophores.
The detection of different substances in one and the same specimen usually involves the use of distinct fluorophores, which have respective excitation and emission wavelengths. Light sources with different spectra of emission are hence used in succession to analyse the responses in the excitation and emission bands of each fluorophore.
A limitation of the known systems lies in the difficulty of illuminating in a uniform way the portion of the support in which the material to be analysed is contained. Frequently, in fact, the supports comprise plates in which wells are made orderly arranged to form a matrix array. The supports are loaded in lightproof chambers of analyser apparatuses for being read. On account of constructional constraints of the sources and of the analyser apparatuses, the area of the light source, for example a LED, its directionality and its distance from the support, the entire array is not illuminated uniformly, resulting in variations of incident optical power from one well to the other. Since the fluorophores are excited by the incident optical power, their response can be affected by the non-uniform illumination.
On the other hand, using a number of light sources with one and the same nominal spectrum of emission would not prevent variations of illumination due to process dispersions.
It should moreover be considered that the optical power supplied by the light source is not constant over time, but varies for example in proportion to the ambient temperature and can undergo modifications due to ageing of the component.
Hence, the non-uniform and non-constant illumination can lead to unreliable readings.